Cytokines are secreted proteins that regulate cell growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, regulating lymphoid development and differentiation. Cytokines also regulate immune homeostasis, tolerance, and memory. Not surprisingly, cytokines are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and psoriasis. Understanding the molecular basis of cytokine action provides important insights into the pathogenesis of immune-mediated disease and offers new therapeutic targets. Cytokine receptors are associated with Janus family kinases (Jaks), which initiate signaling (see project AR041106-14). Following activation of Jaks, the next step in signaling is the activation of a family of transcription factors called Stats (signal transducers and activators of transcription) (see project AR041159-01). To better understand the molecular actions of cytokines, we performed transcriptional profiling of mouse and human T cells activated by interleukin (IL)-12. We identified a large number of inducible genes, some of which had been previously recognized as being IL-12-inducible. One such gene was the gene encoding the serine/threonine kinase Cot/Tpl2. This kinase was directly inducible by IL-12 and inhibited by IL-4. As IL-12 activates the transcription factor Stat4, we also assessed the dependence of inducible MAP3K8 expression in Stat4-deficient mice. Furthermore, using chromatin immunoprecipitation, we found that MAP3K8 was a direct Stat4 target. To define the role of MAP3K8, we studied MAP3K8 knockout mice. We found that Thelper1 differentiation of MAP3K8-/- mice was markedly deficient. We further found that this was associated with failure to upregulate the transcription factors Stat4 and Tbox21 (T-bet), thus explaining the failure to properly generate IFNg. Challenge of MAP3K8-deficient mice with Toxoplasma gondii showed that these mice had increased susceptibility to this model pathogen, consistent with the impaired ability to generate IFNg. Conversely, in the model of autoimmune disease that mimics multiple sclerosis, experimental autoimmune encephalomyelitis or EAE, MAP3K8-knockout mice had reduced severity of disease. We further established that OVA-immunized Tpl2(-/-) mice express high levels of IgE and develop more severe bronchoalveolar eosinophilic inflammation than Tpl2(+/+) controls, when challenged with OVA intranasally. Bronchoalveolar exudates and supernatants of OVA-stimulated splenocytes from immunized Tpl2(-/-) mice express elevated levels of IL-4 and IL-5, suggesting that Tpl2 ablation promotes the Th2 polarization of the T cell response. Anti-CD3 stimulation of CD4(+) T cells of wild-type and Tpl2 knockout mice revealed that Tpl2 ablation gives rise to a cell autonomous T cell defect that is primarily responsible for the Th2 polarization of the T cell response to Ag. This observation was further supported by experiments addressing the expression of Th1 and Th2 cytokines in OVA-stimulated mixed cultures of CD4(+) T cells from Tpl2(+/+)/OT2 or Tpl2(-/-)/OT2 mice and dendritic cells from Tpl2(+/+) or Tpl2(-/-) mice. Further studies revealed that Th1 cells express significantly higher levels of Tpl2 than Th2 cells. As a result, Tpl2(-/-) Th1 cells exhibit a stronger defect in ERK activation by anti-CD3 than Th2 cells and express low levels of T-bet. Given that the development of Th1 and Th2 cells depends on positive feedback signals from the T cells, themselves, the functional defect of the Tpl2(-/-) Th1 cells provides a mechanistic explanation for the T cell autonomous Th2 polarization in Tpl2(-/-) mice. We also investigated the role of MAP3K8 in innate immune cells. To address this issue, we infected Tpl2(-/-) mice with the model pathogen Listeria monocytogenes. We found that Tpl2(-/-) mice infected i.v. with L. monocytogenes had increased pathogen burdens compared with wild-type mice and rapidly succumbed to infection. Enhanced susceptibility correlated with impaired signaling through TLR2 and nucleotide-binding oligomerization domain 2, two receptors previously shown to mediate Listeria recognition. Surprisingly, TNF production in response to infection was not significantly impaired, even though Tpl2 has been implicated in the regulation of TNF. We found that the role of Tpl2 has cell-type specific effects in regulating TNF and transduces signals from some, but not all, pattern recognition receptors (PRR). In contrast to the cell-type- and receptor-specific regulation of TNF, we found that Tpl2 is essential for IL-1beta production from both macrophages and dendritic cells. These studies implicate Tpl2 as an important mediator for collaboration of pattern recognition receptors with danger-associated molecular patterns to induce TNF and IL-1beta production and optimal host defense.